| Part ?. The Influence of Periplaneta americana Extracts on Intestinal Microflora in Ulcerative Colitis RatsObjective: To investigate the effects of Periplaneta americana extracts YS-1 and YS-2 of different proportions on intestinal microflora of rats with ulcerative colitis(UC) induced by xenogenic antigen combined with acetic acid.Methods: UC rats induced by acetic acid and xenoantigen were treated with YS-A\B\C composed of different proportions of YS-1 and YS-2. PCR primers were designed according to the specific DNA sequence of bacterial species, to construct a standard of recombinant plasmid containing specific amplified fragments. A quantitative real-time PCR assay based on SYBR Green I dye was established for the determination of total intestinal bacteria, Bacteroides, Bifidobacterium, Lactobacillus,Escherichia coli,Enterococcus and Sulfate-reducing bacteria of the stool samples from UC rats in the model and administration groups. Statistical analyses were carried out both on absolute quantification and flora percentage changes.Results: 1. A quantitative real-time PCR assay was established for the determination of total intestinal bacteria, Bacteroides, Bifidobacterium, Lactobacillus, Escherichia coli, Enterococcus and Sulfate-reducing bacteria.2. The populations of Bifidobacterium, Lactobacillus in feces of the UC model group (4.13±0.28, 4.48±0.32,log bacteria copy number,the same as below) were significantly lower than those (4.92±0.30.5.13±0.67) in the normal control group(P<0.05). The populations of Escherichia coli and Enterococcus (3.14±0.46,2.16±0.35) were decreased compared with the normal control group (3.76±0.54,2.40±0.39) without statistical significance, and those of Bacteroides and Sulfate-reducing Bacteria (5.75±0.38, 4.22±0.18) displayed an increasing trend compared with normal control group (5.46±0.25, 3.77±0.21). There was no significant difference in the total number of intestinal flora (7.05±0.13) compared with the normal control group (7.02±0.15).3. After treatment, the populations of Bifidobacteria of YS-A (YS-1/YS-2=2:1)YS-C (YS-1/YS-2=1:2) at all dose groups and those of YS-B (YS-1/YS-2=1:1) at middle, high dose groups increased to a certain extent compared with the model group.The populations of Bifidobacterium in the model group and administration groups were significantly lower than those in the normal group (P<0.05, P<0.01), while increasing trends could be found for various dose groups of YS-A\B\C than the model group.4. The number of Lactobacillus in YS-B low dose group was significantly higher than that in model group (P<0.01). The number of Lactobacillus in SASP group,YS-A middle and high dose group, YS-B middle dose group, and YS-C low dose group showed increasing trends. The number of lactobacilli in YS-B high dose group decreased compared with other groups without statistical significance.5. The populations of Bacteroides of YS-B at middle and high dose groups were significantly lower than that at model group (P<0.05). The numbers of Bacteroides of SASP and YS-C at low dose group were lower than that of model group. The population of Bacteroides of YS-A at middle group accounted for a remarkable increase in the normal group.6. The numbers of Escherichia coli of YS-B low, middle dose groups and YS-C middle, high dose groups were significantly higher than the model group (P<0.05),while those of SASP group and YS-C low dose group possessed increasing trends compared to the model group.7. The numbers of Enterococcus of YS-A low and medium dose group, YS-B high dose group exhibited a downward trend compared with other groups. The numbers and proportions of Enterococci in the other groups were higher than those in the model group.8. YS-A and YS-B of all dose groups could significantly reduce the number of Sulfate-reducing bacteria (P<0.05. P<0.01). The number of Sulfate-reducing bacteria in SASP and YS-C groups decreased compared with the model group.9. The number of total bacterial flora in YS-A was significantly lower than those in normal group and model group (P<0.05). The others were not significantly different with one another.10. The results of analysis on flora percentage changes of intestinal microflora species in individual samples were consistent with their corresponding absolute quantitative analyses.Conclusions: 1. SYBR Green I absolute quantitative real-time PCR method was established to detect six kinds of intestinal microflora tightly correlated to UC as well as the total bacterial flora. The method was feasible to analyse the content changes of these six dominant bacteria species in the feces of UC rats in the model and administration groups.2. Obvious imbalance was detected for the intestinal microflora of rats with UC induced by acetic acid and xenoantigen. The populations of Bifidobacterium,Lactobacillus, Escherichia coli and Enterococcus in feces of UC model group were significantly lower than those of the normal control group, while those of Bacteroides and Sulfate-Reducing Bacteria were significantly higher. The numbers of total flora in feces of UC rats were basically constant, and the changes of the relative proportions of intestinal flora were consistent with the results of absolute quantitative analyses.3. The results suggested that YS-A, YS-B and YS-C could adjust the intestinal micro-ecological environment to a certain extent, and balance the intestinal flora populations, contributing to the development of new drugs in clinical treatment of UC.Part II. Study of Preliminary Mechanism on Anti-tumor Activity of Insect ExtractsObjective: To discover the anti-tumor bioactive extracts from insects by in vitro screening assays and to preliminarily study on the mechanism of action.Methods: A549 cells, Hela cells and Bel-7402 cells were incubated respective with 350 extracts from insects for 48 hours, the inhibitory effects of cell proliferation in different concentrations were determined by MTT assay and IC50 values were calculated accordingly. The time-effect and dose-effect relationships were further determined for active samples worthy of further study. Flow cytometry was used for the preliminary study on the mechanism of proliferation inhibition on tumor cells.Apoptosis and necrosis were detected by Annexin V-FITC/PI double stainings; cell cycle distribution was detected by PI staining; the changes of mitochondrial membrane potential were detected by JC-1 staining.Results: 1.31 samples with interesting cytotoxic activity were screened out from 350 insect-origin extracts. Among them, 10, 10, 22 samples exhibited certain proliferation inhibitory activity on A549 cells, Hela cells, Bel-7402 cells, respectively.2. The extract (YS-122) with relatively satisfactory inhibitory effect on all the three human tumor cell lines was selected for further study on the time-effect and dose-effect relationships. YS-122 demonstrated promising inhibitory effects on Hela,A549, Bel-7402 cells at 24 h, 48 h and 72 h, and with a dose-dependent manner. The IC50 value of 72 h towards Hela cell line was 4.96; The IC50 values of 48 h and 72 h towards A549 cells were 31.64 and 6.93; The IC50 values of 48 h and 72 h against Bel-7402 cells were 32.72 and 4.06.3. Annexin V-FITC/PI double staining method was used to analyze the effect of sample (YS-122) on apoptosis and necrosis of the three human tumor cells. After different concentrations of YS-122 applied to tumor cells, the proportion of early apoptosis,late apoptosis and early necrosis,late necrosis cells showed an upward trend compared with the negative control group, and the difference was statistically significant. With the same concentration of YS-122 treated Hela cells, the proportions of early apoptosis, late apoptosis, early necrosis, late necrosis cells did not change much at 24 h and 48 h, the proportions of late apoptosis and early necrosis cells at 72 h increased significantly.4. PI staining method was used to analyze the effect of sample (YS-122) on cell cycle distribution of the three human tumor cells. After different concentrations of YS-122 applied to tumor cells for 24 h, 48 h and 72 h, the proportions of Sub(G0/G1),G0/G1, S,G2,M, S+G2/M peaks were significantly different from those of negative control group. At the same action time, the proportions of Sub(G0/G1) peak and S peak increased and that of G0/G1 peak decreased when the concentrations of YS-122 increased, the differences were statistically significant (P<0.01).5. JC-1 staining was used to analyze the effect of YS-122 on the mitochondrial membrane potential of the three human tumor cells. After different concentrations of YS-122 applied to the tumor cell lines for 48 h, the mitochondrial membrane potentials were all decreased, which were significantly different from those of negative control group (P<0.01).Conclusion: 1.31 samples with remarkable cytotoxic activity were screened out from 350 insect-origin extracts.2. YS-122 possessed a satisfactory inhibitory effects towards the three human tumor cells (cisplatin as positive control), and also had good time-dependent and dose-dependent relationships.3. The capability of YS-122 in reducing the proliferation of tumor cells might be due to its activating apoptotic signals of mitochondrial pathways, stimulating tumor cell's cycle arresting in S phase, blocking the progress of tumor cells from S phase to G2/M phase, blocking cell mitosis, and promoting cell apoptosis in G1 phase, thereby reducing the proliferation of tumor cells. |
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